PpbHLH14.1-PpCCD4a regulatory module modulates lutein accumulation and peel colouration in pear (Pyrus pyrifolia)

Lutein is a nutritionally important carotenoid that contributes to the peel colour and fruit quality in pear. However, the molecular mechanisms regulating lutein accumulation remain poorly understood. In this study, we identified carotenoid cleavage dioxygenase 4a (PpCCD4a) as a key factor associated with variation in lutein content in pear (Pyrus pyrifolia) fruit peel. Gene expression profiling and carotenoid analysis across multiple cultivars and fruit developmental stages revealed a strong negative relationship between PpCCD4a transcript levels and lutein accumulation. Functional analyses demonstrated that transient overexpression of PpCCD4a reduced lutein content and attenuated yellow peel colouration, whereas gene silencing resulted in increased lutein accumulation and enhanced pigmentation. Furthermore, a basic helix-loop-helix transcription factor, PpbHLH14.1, was identified as an upstream regulator of PpCCD4a. Dual-luciferase assays showed that PpbHLH14.1 activates the PpCCD4a promoter, and yeast one-hybrid assays confirmed its direct binding to E-box motifs within the promoter region. Manipulation of PpbHLH14.1 expression led to coordinated changes in PpCCD4a transcript levels, lutein accumulation, and peel colouration, supporting a regulatory relationship between these factors. Collectively, these results establish a PpbHLH14.1-PpCCD4a regulatory pathway that modulates lutein accumulation in pear fruit. This study provides new insights into the transcriptional regulation of carotenoid degradation and identifies potential targets for improving fruit nutritional and visual quality.